Ion channels are the major determinant for excitability changes of neurons. Our long-term objective is to elucidate the mechanism by which slow excitation and slow inhibition of brain neurons take place. These events occur on time scales of tens of seconds to a few minutes, and are mostly mediated by G proteins. Our immediate research will focus on clarifying the signal transduction mechanism by which G-protein-coupled inward rectifier Kv (GIRK) channels are modulated by substance P (SP), an excitatory peptide neurotransmitter. Native cultured noradrenergic neurons in the locus coeruleus (LC) from newborn rats and mice, as well ascloned GIRKs expressed in HEK293 cells will be used. Electrophysiological and molecular biological techniques will be employed for the investigation. The LC contains neurons that innervate and supply norepinephrine to a wide area of the brain, and plays a vital role in arousal and alertness. Furthermore, LC neurons often degenerate in Alzheimer's disease. LC neurons are dually regulated by opposing signals: inhibitory transmitters, such as somatostatin, activate GIRK channels, whereas excitatory transmitters, such as SP, inhibit the GIRK activity that is activated by somatostatin. The mechanism of the GIRK inhibition, however, is controversial and yet to be determined. The goal of the proposed project is to elucidate the signal transduction mechanism of the SP-induced GIRK channel inhibition. Three possible signal transduction pathways could be responsible for the SP-induced GIRK channel inhibition: the phosphatidyl inositol 4,5-bisphosphate (PIP2) pathway, the protein kinase C pathway, and a new pathway, which we designate as the "quick pathway." The existence of the quick pathway is hypothesized because the inhibition is too rapid to be accounted for by the other pathways. We intend to elucidate the mechanism of this pathway. We also intend to investigate the possible synergistic relation between the quick and the PIP2 pathways.